Multiple antibiotic resistance is becoming a more frequent observation in clinical isolates throughout the world. A chromosomal regulatory locus called mar in E. coli and other enterobacteriaceae confers resistance to a variety of antibiotics when overexpressed. This kind of resistance has been found in E. coli clinical isolates from several countries. The proposed studies are designed to purify and define functionally the regulatory proteins of two divergent operons in the mar locus and to determine genes in the mar regulon that are targets of these proteins and that produce the Mar phenotype. The marRAB operon encodes MarR, a repressor of transcription, and MarA and MarB, putative activators of distant loci. Component genes will be cloned, purified as fusion proteins and tested for function. Electrophoresis will assist in identifying proteins involved in the Mar system. Potential DNA sequences for the regulatory proteins will be identified. Undefined resistance mechanisms for chloramphenicol, beta-lactam antibiotics and rifampicin will be studied. The frequency of mar operon mutations will be examined, as will the possible existence of similar systems in Pseudomonas aeruginosa. Homologous loci in other chromosomally resistant organisms such as the mycobacteria will be sought.